Specific aim 1: Develop and use in vitro 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural system development and degeneration. An important aspect of neuronal system development is the influence of endothelial cells which are part of the blood brain barrier and interact with adjacent neurons by releasing growth factors and cytokines. We have used our human CD34-derived neural stem cells to differentiate oligodendrocyte progenitor cells (OPC). We found that vascular endothelial growth factor -A (VEGF-A) increases proliferation of OPC. This finding has been accepted by Glia for publication. While the finding implies a close interaction in the development of neural system and endothelial cells, it is difficult to generate a 3D model with both neuronal cells and endothelial cells using iPSC without derive the cells separately at first, as they are from different germ layers. We set to overcome the technical hurdle and successfully generated a 3D organoid model using a single embryoid body generated from iPSC. The 3D organoid was positive when immunostained for CD31, an endothelial marker and MAP2 and beta-III tubulin, markers for neurons. Other genes such as VEGF-A and TIMP-2 were also expressed at high level when detected by RT-PCR, suggesting an environment supportive of spontaneous development of vascular endothelial cells and neural cells. We are using this model to study the possible involvement of endothelial cells in motor neuron disorders such primary lateral sclerosis (PLS) and ALS. We found that using brain organoids, both ALS and PLS showed delayed neuronal differentiation compared to their normal aged controls. We are in the process to expend our project to include more samples and also with different genomic backgrounds. The finding has been presented on the ISSCR annual meeting at Melbourne, Australia, 2018 Specific aim 2: Study the role of HERV-K on human neural development. We have found that human endogenous retroviruses K (HERV-K) is expressed on human iPSC. Inhibition of HERV-K Env protein enhanced neuronal differentiation, indicating HERV-K plays a role in keep the stem cells from differentiation. We determined that also Ch22 is the most activated loci for HERV-K env in human iPSCs. Loci in Ch12 and Ch19 are the ones that produce Env proteins. In regard of the molecular pathways underlying the effect of HERV-K Env on the iPSC maintenance and function, we combined the data from RNAseq, Mass spec and found mTOR pathway was highly involved. Using real-time PCR and Western-blot assay, we confirmed that HERV-K Env interacts with CD98HC to activate mTOR pathway, which in turn activated LPCAT1, an important enzyme for lipid synthesize and likely plays a role in tumor genesis. The result of mTOR pathway activation is responsible for the HERV-K Env regulated morphological changes in iPSC. We have prepared the manuscript on the finding. Specific aim 3: Developed an in vitro HERV-K transmissible model using rhesus macaque cells. It has been reported that brain tissues from ALS may contain infectious agents which when transplanted can cause rhesus macaques to show similar symptoms and etiology. While it is difficult to study the possibility that HERV-K is the agent that transmitted the disorder in vivo, it is possible to study the possibility using in vitro cell cultures first. We have been collaborating with Dr. John Heiss to derive cells form rhesus macaques. We have cultured fibroblasts from rhesus skin samples and is in the process of generating neural stem cells from them. The goal is to infect rhesus cells with HERV-K plasmid and study its transmissible capability and its effect on neuronal cells. Specific aim 4: facilitate the research and therapeutic developments for neurological disorders using our models and methods. We are in collaboration with other investigators by providing material support and training of the iNSC/iPSC generation. We provided iPSC 3D organoid generation training to Dr. Pankaj Seth. We helped Dr. Henry Levis lab with technique support, cell lines and expertise on developing a project studying the retroelements in neurological disorders using neural stem cells. The project has resulted in preliminary result and a meeting abstract. We also provide motor neurons and collaborated with Dr. Sergey Iordanskiy on a project studying the effect of radiation on the expression of HERV-K in human cells. Preliminary result showed that radiation increased HERV-K expression in monocytes and which may cause neuronal toxicity. The preliminary also resulted in a meeting abstract. We will continue the fruitful collaboration which will likely provide novel insights of pathologies o neurological disorders. Bibliography in press: 1.Katherine Roche, Michael Bemben, Thien Nguyen, Yan Li, Tongguang Wang, and Roger Nicoll Isoform-specific cleavage of neuroligin-3 reduces synapse strength. (2018) Molecular Psychiatry, in press. 2.Elliot Choi, Yadi Xu, Marie Medynets, Maria Chiara G. Monaco, Eugene O. Major, Avindra Nath and Tongguang Wang. Activated T cells induce proliferation of oligodendrocyte progenitor cells via release of vascular endothelial cell growth factor-A. (2018) Glia, in press. Meeting abstracts: 3.Lechsinska A, Ferreira-Worman Z, Payer L, Wang T, Li W, Burns K, Nath A, and Levin H. The Role of Transposable Elements in Neurological Disorders. Cold Spring Harbor Laboratory Conference on Transposable Elements 2018. 4.Natallia Mikhalkevich, Rok Tkavc, Marie Medynets, Tongguang Wang, Avindra Nath and Sergey Iordanskiy. Human endogenous retrovirus type K (HERV-K) is involved in inflammatory response to gamma-radiation. USU Research Days 2018 5.Tongguang Wang, Marie Medynets, Mary Kay Floeter, Avindra Nath. In vitro modeling of motor neuron disorders using iPSCs suggests unique property of primary lateral sclerosis. ISSCR Annual meeting, Melbourne, Australia 2018, June. Student Mentoring: Ms. Anna Bagnell joined our lab as a summer student intern. She received the training on the 3D organoid cultures and completed the internship successfully with a NINDS exceptional summer student award.